Plants constantly suffer from stresses even in a normal growth environment. Such stresses variously include salt, drying, high temperature, low temperature, intense light, air pollution, and the like. Salt stress has received attention in terms of agricultural production. When soils and stones are decomposed, salts are generated, and the generation of salts is constantly continued. When it rains, the salts may flow into the river or the sea. In desert areas of low rainfall, a lesser amount of salts flow out, so that the salt concentration is considerably higher in soil water. Plants draw salts (nutrients) along with water osmotically through roots. When the salt concentration is high, plants cannot draw water. Moreover, the growth of the plants is inhibited due to physiological actions specific to ions. It is known that responses of plants to a salt stress overlap responses to environmental stresses, such as drying, high osmotic pressure, low temperature, and the like. These stresses lead to considerably severe damage to agriculture.
Recently, due to use of fertilizers in bulk or long-term sequential cropping, it is often observed that a high concentration of salt is accumulated in soil. Especially in greenhouse soil, detrimental salt accumulation frequently occurs. In areas near seashores, sea water or sea breeze causes damage. In arid or semiarid regions, salts are accumulated in the surface layer of soil due to excessive evaporation. These problems limit use of agricultural lands. In order to solve such problems, generally, the affected soil is exchanged or salts are removed by irrigation. However, these methods require huge expense or effort. The removal of salts by irrigation causes a large volume of salt water to flow into surrounding regions, leading to environmental pollution. Restriction of irrigation is now under consideration.
Therefore, it is very important to find a plant tolerant to such a salt stress.
According to studies for salt tolerant plants, which have been carried out at home and abroad, it is known that when salt tolerant plants are transferred from under non-stress conditions to salt stress conditions, expression of new genes is induced and the products of these genes play a role in salt stress tolerance. It is known that among plants of the genus Nicotiana, there are some types of plant having salt stress tolerance. Isolation of a relevant gene has been reported (Nelson et al., (1992) Plant Molecular Biology, vol. 19, 577–588; Yun et al. (1996) Plant Physiology, vol. 111, 1219–1225). Another exemplary gene of plants of the genus Nicotiana relating to response to a salt stress is a gene derived from Nicotiana paniculata of a type having salt stress tolerance, which is described in Japanese Laid-Open Publication No. 11-187877, Japanese Laid-Open Publication No. 11-187878, Japanese Laid-Open Publication No. 11-187879, and Japanese Laid-Open Publication No. 11-187880. Japanese Laid-Open Publication No. 11-187877 describes a novel gene induced by salt stress. A gene product encoded by this gene is considered to have a function of conferring moisture stress tolerance to plants. Japanese Laid-Open Publication No. 11-187878 describes a novel potassium channel gene induced by salt stress. The potassium channel gene encoded by this gene is considered to have a function of conferring moisture stress tolerance to plants. Japanese Laid-Open Publication No. 11-187879 describes a novel INPS gene induced by salt stress. The INPS gene encoded by this gene has a function of conferring moisture stress tolerance to plants. Japanese Laid-Open Publication No. 11-187880 describes a novel chloroplast type fructose bisphosphate aldolase induced by salt stress. The aldolase encoded by this gene is considered to have a function of conferring moisture stress tolerance to plants.
Two genes relating to response to salt stress in rice are chloroplast glutamine synthetic enzyme (GS2) gene (Hoshida et al., Plant Mol. Biol. 43:103–11(2000)); and Δ1-pyrroline-5-carboxylate (P5C) synthetic enzyme (OsP5CS) gene (Igarashi et al., Plant Mol. Biol. 33:857–65(1997)). In the above-mentioned literature, it is described that the GS2 gene enhances photorespiration ability so that salt stress tolerance is conferred to a plant. A gene for OsP5CS involved in biosynthesis of proline is induced by a high concentration of salt, dehydration, abscisic acid treatment, and low temperature. Expression of the OsP5CS gene under salt stress is stably increased in a salt tolerant cultivar Dee-gee-woo-gen, while it is slightly increased in salt sensitive bred variety IR28.
A number of gene disruption strains of rice have been produced by the property of rice retrotransposon Tos17 that it is activated by culture to undergo transposition. Transposons are mutagenic genes which are ubiquitous in the genomes of animals, yeast, bacteria, and plants. Transposons are classified into two categories according to their transposition mechanism. Transposons of class II undergo transposition in the form of DNA without replication. Examples of class II transposons include Ac/Ds, Spm/dSpm and Mu elements of maize (Zea mays) (Fedoroff, 1989, Cell 56, 181–191; Fedoroff et al., 1983, Cell 35, 235–242; Schiefelbein et al., 1985, Proc. Natl. Acad. Sci. USA 82, 4783–4787), and Tam element of Antirrhinum (Antirrhinum majus) (Bonas et al., 1984, EMBO J, 3, 1015–1019). Class II transposons are widely used for gene isolation by means of transposon tagging. Such a technique utilizes a property of transposons, that is, a transposon transposes within a genome and enters a certain gene and, as a result, such a gene is physiologically and morphologically modified, whereby the phenotype controlled by the gene is changed. If such a phenotype change can be detected, the affected gene may be isolated (Bancroft et al., 1993, The Plant Cell, 5, 631–638; Colasanti et al., 1998, Cell, 93, 593–603; Gray et al., 1997, Cell, 89, 25–31; Keddie et al., 1998, The Plant Cell, 10, 877–887; and Whitham et al., 1994, Cell, 78, 1101–1115).
Transposons of class I are also called retrotransposons. Retrotransposons undergo replicative transposition through RNA as an intermediate. A class I transposon was originally identified and characterized in Drosophila and yeast. A recent study has revealed that retrotransposons are ubiquitous and dominant in plant genomes (Bennetzen, 1996, Trends Microbiolo., 4, 347–353; Voytas, 1996, Science, 274, 737–738). It appears that most retrotransposons are an integratable but non-transposable unit. Recently, it has been reported that some retrotransposons of such a type are activated under stress conditions, such as injury, pathogen attack, and cell culture (Grandbastien, 1998, Trends in Plant Science, 3, 181–187; Wessler, 1996, Curr. Biol., 6, 959–961; Wessler et al., 1995, Curr. Opin. Genet. Devel., 5, 814–821). For example, such activation under stress conditions was found in retrotransposons of tobacco, Tnt1A and Tto1 (Pouteau et al., 1994, Plant J., 5, 535–542; Takeda et al., 1988, Plant Mol. Biol., 36, 365–376), and a retrotransposon of rice, Tos17 (Hirochika et al., 1996, Proc. Natl. Acad. Sci. USA, 93, 7783–7788).
The rice retrotransposon Tos17 is a class I element in plants which has been extensively studied. Tos17 was cloned by RT-PCR using degenerate primers which had been prepared based on a conserved amino acid sequence of the reverse transcriptase domains of Ty1-copia group retro-elements (Hirochika et al., 1992, Mol. Gen. Genet., 233, 209–216). Tos17 has a length of 4.3 kb and has two identical LTRs (long terminal repeats) of 138 bp and a PBS (primer binding site) which is complementary to the 3′ end of the initiator methionine tRNA (Hirochika et al., 1996, supra). Transcription of Tos17 is strongly activated by tissue culture, and the copy number of Tos17 increases with time in culture. Its initial copy number in Nipponbare (a Japonica variety), which is used as a genome research model, is two. In plants regenerated from tissue culture, its copy number is increased to 5 to 30 (Hirochika et al., 1996, supra). Unlike class I transposons found in yeast and Drosophila, Tos17 undergoes random transposition in a chromosome and induces mutation in a stable manner. Therefore, Tos17 provides a useful tool in reverse genetics for analyzing the function of a gene in rice (Hirochika, 1997, Plant Mol. Biol. 35, 231–240; K. Shimamoto Ed., 1999, Molecular Biology of Rice, Springer-Verlag, 43–58).